Toxoplasma gondii, is an obligate intracellular parasite known to chronically infect a third of the human population and the cause of devastating disease in immunocompromised individuals and in those infected congenitally. Toxoplasma is considered a neglected parasitic disease of the United States by the Center for Disease Control and there is a dire need for new and effective therapeutics. Critical to the discovery of new drug targets is the characterization of events and proteins that are essential and unique to the parasite. Events required for Toxoplasma survival such as invasion and egress are regulated by calcium signaling processes that include proteins unique to the parasite. Interestingly, Toxoplasma egress depends on calcium signaling in both the parasite and host cell. How calcium fluxes and signaling within the parasite relate and translate to those occurring in the host and vice versa is not understood. While inside its host cell Toxoplasma divides within a specialized parasitophorous vacuole (PV) that protects it from cellular clearance mechanisms and provides it with the required nutrition. We hypothesize that, given its position at the interface between parasite and host, the PV also plays a critical role in integrating the signaling events that control egress and other calcium dependent events. To test this hypothesis we are combining a gene candidate approach with a novel screen for PV signaling proteins. Using a bioinformatics approach we have identified two secreted proteins that contain EF hand domains, which are found in numerous calcium signaling proteins including calmodulin and the parasite specific calcium dependent protein kinases. Interestingly, both of these proteins localize to the PV. Our first aim consists of determining the function of these two putative calcium-binding proteins during the lytic cycle of the parasite. Our second aim will be to identify and characterize signaling proteins associated with the PV. The full complement of PV proteins is far from known given significant challenges in separating its content from that of the parasite and host cell. Our novel approach to determine the protein makeup of the PV will be to identify proteins that interact or are near known vacuolar proteins using the proximity based BioID protein-protein interaction trap. Once we have identified putative novel PV proteins we will characterize six of them focusing on those likely to be involved in signaling events. In conjunction, our results will shed light on how host and parasite signaling pathways are integrated and exploit the essential roles of the PV and egress to discover new drug targets for this important and neglected parasite.